Improved gene delivery technologies are needed for the treatment of disease in animals. Many diseases and conditions can be treated with gene-delivery technologies, which provide a gene of interest to an animal suffering from the disease or the condition. An example of such disease is Type 1 diabetes. Type 1 diabetes is an autoimmune disease that ultimately results in destruction of the insulin producing β-cells in the pancreas. Although animals with Type 1 diabetes may be treated adequately with insulin injections or insulin pumps, these therapies are only partially effective. In addition, hyper- and hypoglycemia occurs frequently despite intensive home blood glucose monitoring. Finally, careful dietary constraints are needed to maintain an adequate ratio of calories consumed. Development of gene therapies providing delivery of the insulin gene into the pancreas of diabetic animals could overcome many of these problems and result in improved life expectancy and quality of life.
Several of the prior art gene delivery technologies employed viruses that are associated with potentially undesirable side effects and safety concerns. The majority of current gene-delivery technologies useful for gene therapy rely on virus-based delivery vectors, such as adeno and adeno-associated viruses, retroviruses, and other viruses, which have been attenuated to no longer replicate. (Kay, M. A., et al. 2001. Nature Medicine 7:33-40).
There are multiple problems associated with the use of viral vectors. First, they are not tissue-specific. In fact, a gene therapy trial using adenovirus was recently halted because the vector was present in a patient's sperm (Gene trial to proceed despite fears that therapy could change child's genetic makeup. The New York Times, Dec. 23, 2001). Second, viral vectors are likely to be transiently incorporated, which necessitates re-treating a patient at specified time intervals. (Kay, M. A., et al. 2001. Nature Medicine 7:33-40). Third, there is a concern that a viral-based vector could revert to its virulent form and cause disease. Fourth, viral-based vectors require a dividing cell for stable integration. Fifth, viral-based vectors indiscriminately integrate into various cells, which can result in undesirable germline integration. Sixth, the required high titers needed to achieve the desired effect have resulted in the death of one patient and they are believed to be responsible for induction of cancer in a separate study. (Science, News of the Week, Oct. 4, 2002).
Accordingly, what is needed is a new method to produce transgenic animals and humans with stably incorporated genes, in which the vector containing those genes does not cause disease or other unwanted side effects. There is also a need for DNA constructs that would be stably incorporated into the tissues and cells of animals and humans, including cells in the resting state that are not replicating. There is a further recognized need in the art for DNA constructs capable of delivering genes to specific tissues and cells of animals and humans and for producing proteins in those animals and humans.
When incorporating a gene of interest into an animal or human for the production of a desired protein or when incorporating a gene of interest in an animal for the treatment of a disease, it is often desirable to selectively activate incorporated genes using inducible promoters. These inducible promoters are regulated by substances either produced or recognized by the transcription control elements within the cell in which the gene is incorporated. In many instances, control of gene expression is desired in transgenic animals and humans so that incorporated genes are selectively activated at desired times and/or under the influence of specific substances. Accordingly, what is needed is a means to selectively activate genes introduced into the genome of cells of a transgenic animal or human. This can be taken a step further to cause incorporation to be cell-specific, which prevents widespread gene incorporation throughout the body. This decreases the amount of DNA needed for a treatment, decreases the chance of incorporation in gametes, and targets gene delivery, incorporation, and expression to the desired tissue where the gene is needed to function.
RNAi has been targeted as a tool for several uses including treatment of genetic abnormalities and disease, cancer, and development. There are mainly two types of short RNAs that target complementary messengers in animals: small interfering RNAs and micro-RNAs. Both are produced by the cleavage of double-stranded RNA precursors by Dicer, a member of the Rnase III family of double-stranded specific endonucleases, and both guide the RNA-induced silencing complex to cleave specifically RNAs sharing sequence identity with them. RNAi technology can be used in therapeutic approaches to treat disease and various conditions. However, a major drawback to RNAi therapy has been the lack of a reliable delivery method of the short RNA sequences. Most researchers working in the field rely on producing short double stranded RNA (dsRNA) in the laboratory and then delivering these short dsRNAs either by direct injection, electroporation, by complexing with a transfecting reagent, etc. The result is gene silencing, but only as long as the dsRNA remains present in the cell, which generally begins to decrease after about 20 h. In order to obtain lasting therapeutic effects, the RNAi sequence must be expressed long term, preferably under a constitutive promoter. In order to accomplish RNAi expression in a plasmid-based vector and subsequent recognition by RNA induced silencing complex (RISC), the RNA must be double stranded. To obtain dsRNA from a vector, it must be expressed as a short hairpin RNA (shRNA), in which there is a sense strand, a hairpin loop region and an antisense strand (M. Izquierdo. 2004. Short interfering RNAs as a tool for cancer gene therapy. Cancer Gene Therapy pp 1-11; Miyagishi et al. 2004. J Gene Med 6:715-723). The hairpin region allows the antisense strand to loop back and bind to the complimentary sense strand.